Norleucine is an analog of the amino acid methionine that can be misincorporated into a protein in the place of methionine. In Escherichia coli (E. coli) norleucine can be biosynthesized by the enzymes of the leucine biosynthetic pathway. When expressed in E. coli many heterologous proteins have norleucine mistakenly incorporated in places methionine residues should appear. The misincorporation of norleucine is undesirable because it usually results in the production of an altered protein, having less than optimal characteristics.
The amino acid norleucine (2-aminocaproic acid; 2-aminohexanoic acid; see FIG. 1), first known to science from synthetic preparations made in 1870, attracted great interest after being claimed in 1882 by the chemist Ludwig Thudichum to have been found as one of the natural amino acids of proteins. Other workers seemed to confirm this finding, claiming in 1912-1913 to have found norleucine in proteins. These observations were ostensibly confirmed and extended by yet more laboratories during the following two decades. This body of literature was reviewed by Schmidt (1933), and led him to recommend that norleucine be added to the list of accepted constituent amino acids of proteins. However, within 12 years, it was conclusively shown that the analytical techniques employed by the earlier workers had misled them, and that norleucine did not naturally occur in proteins (Consden et al., 1945). The history of norleucine up to 1945, and the error in identifying it as a standard protein amino acid, is recounted in detail by Vickery (1972).
Prior to 1945, while norleucine was still considered to be a standard protein amino acid, nutritional studies with rats demonstrated that, rather than being an essential amino acid, norleucine was actually toxic (Rose, 1938). Norleucine was also shown to be toxic to E. coli and other species of bacteria. It was further observed that the growth inhibition of E. coli by norleucine was reversed by the addition of methionine to the growth medium, thereby establishing that norleucine is an analog of methionine (Harris and Kohn, 1941; Rowley, 1953; Adelberg, 1958; Rowbury, 1965; Karlstrom, 1965).
A review of these and other early reports that norleucine is inhibitory to a variety of species of bacteria is provided by Dittmer (1950). Moreover, Dittmer (1950) noted that norleucine is a structural analog of methionine by virtue of the fact that when the sulfur atom in methionine is replaced by a methylene group norleucine is the result (see FIG. 1). Thus, norleucine was recognized to be an amino acid antagonist, and a structural analog, of methionine. Norleucine attracted significantly more interest than most amino acid analogs, since it was so well characterized and readily available—aspects stemming from the time when norleucine was thought to be a standard protein amino acid.
The first report of the incorporation of exogenously supplied norleucine into protein was that of Rabinowitz et al. in 1954, who observed that exogenous norleucine was incorporated into protein in rat Ehrlich ascites carcinoma cells. A similar observation was made a year later when it was shown that exogenous norleucine could be incorporated into casein in cows (Black and Kleiber, 1955).
These findings were followed, in 1956, by a demonstration that exogenous norleucine was also incorporated into protein by E. coli (Munier and Cohen, 1956). This observation was confirmed by later work (Nisman and Hirsch, 1958), and the phenomenon was also shown to occur in Staphylococcus aureus (Anfinson and Corley, 1969).
Shortly thereafter, it was shown that the incorporation of exogenous norleucine into E. coli protein occurred at the positions where methionine residues normally occurred in the proteins (Cohen and Munier, 1959; Munier and Cohen, 1959; Cowie et al., 1959). This discovery was also confirmed by later work (Neale and Tristam, 1963; Pine, 1967; Kerwar and Weissbach, 1970; Zipori, 1976). The early research into the use of norleucine as an analog of methionine, and its incorporation into protein (when supplied exogenously to a variety of organisms) in place of methionine, was reviewed by Cohen and Gros (1963) and by Meister (1965).
By the mid-1960's it was widely known that exogenously supplied amino acid analogs that are incorporated into protein can have their incorporation blocked by the corresponding natural amino acid, especially when the natural amino acid is present in excess. The literature of that time provides several references establishing this general rule; including those found in Richmond (1962) and Fowden et al. (1967). Within a few years, it was appreciated that for an amino acid analog to be incorporated into protein it must compete with the naturally utilized amino acid for charging onto the corresponding tRNA (Pine, 1978, and Horton and Boime, 1983). These general rules for the incorporation of amino acid analogs into protein were highlighted by specific examples, including that the methionine analog norleucine was blocked from being incorporated into protein by the presence of methionine (Fowden et al., 1967; Pine, 1978; and Barker and Bruton, 1979).
Several studies independently demonstrated that the E. coli methionine-tRNA could be charged with norleucine in vitro and that this aberrant charging was inhibited by methionine (Trupin et al., 1966; Bruton and Hartley, 1968; Lemoine et al., 1968; Old and Jones, 1975; Old and Jones, 1977). Moreover, Old and Jones (1976) found that norleucine inhibited formation of methionyl-tRNA in an E. coli in vitro system; specifically, they showed that the level of methionine charging onto methionine-tRNA decreased gradually with increasing levels of norleucine.
In vivo studies also demonstrated that increased methionine pools reduced the incorporation of norleucine into protein. Fowden et al. (1967), in a review on amino acid analogs and their effects on E. coli and other organisms, stated (at page 91): “A general characteristic of all toxic analogs, whether synthetic or of natural origin, is that their toxic effects are specifically reversed by the normal protein amino acid which is antagonized by the analog”, and (at page 92): “an analog, prior to incorporation into protein, must be activated and transferred to a specific transfer-RNA. The analog therefore must compete with the structurally related protein amino acid at the surface of an aminoacyl-tRNA synthetase”. Fowler (at page 136), referring to the 1964 Ph.D. thesis of S. Neale (University of London), further stated that “the amount of norleucine incorporated into alkaline phosphatase of E. coli K-12 under derepressed conditions was greatly reduced and the abnormally eluting enzyme was not apparent. Incorporation of the analog into the purified enzyme and into gross cell protein was decreased due to increased supplies of intracellular methionine”.
Others have also demonstrated in vivo that low methionine levels typically produce relatively high norleucine incorporation. The level of norleucine incorporated into protein was increased in experiments employing mutants of E. coli unable to make their own methionine, especially when the methionine in the growth medium was exhausted (Yariv and Zipori, 1972; Naider et al., 1972; Brown, 1973). This same observation was made with Staphylococcus aureus (Anfinson and Corley, 1969). Brown (1973) used a mutant of E. coli unable to make its own methionine, grown in a medium containing a high ratio of norleucine to methionine, to prepare proteins with norleucine at the amino-terminus and at internal residues. Barker and Bruton (1979) studied norleucine incorporation into protein in E. coli, reporting in detail on the effects of different ratios of norleucine to methionine on the charging of methionine tRNA with norleucine, and to the subsequent incorporation of norleucine into protein. They demonstrated that the incorporation of norleucine into protein was dependent on the intracellular ratio of norleucine to methionine; significant incorporation of norleucine into protein occurred at a high ratio, and greatly reduced incorporation of norleucine into protein occurred at a low ratio.
It was clear to these workers, as discussed above, that norleucine was not a standard protein amino acid. Indeed, they concluded that norleucine did not even occur in nature as a free amino acid. However, this conclusion was disproved by the observation that Serratia marcescens, an organism closely related to E. coli, is able to biosynthesize norleucine when the leucine biosynthetic system is derepressed (Kisumi et al., 1976, 1977). In this organism, the enzymes of leucine biosynthesis were shown to be responsible for the biosynthesis of the endogenous norleucine. The leucine biosynthetic enzymes have broad substrate specificities (Bogosian et al., 1989), and are capable of forming both leucine and the structurally related norleucine (see FIG. 1). These reports by Kisumi et al. (1976, 1977) represent the first observations of norleucine as a naturally occurring substance.
Thus, by the late 1970's, a great deal was understood about norleucine structure, use, and synthesis. It was clear that norleucine was a structural analog of methionine that could be incorporated into protein by mis-charged methionine-tRNA. Furthermore, it was clear that a sufficient amount of available methionine inhibited the incorporation of norleucine into protein by out-competing norleucine for the charging of methionine-tRNA. Finally, it was known that norleucine was a naturally occurring amino acid, synthesized in bacteria by the enzymes of the leucine biosynthetic pathway.
The stage was thus set for a series of observations made by Bogosian and co-workers in 1985 and published a few years later (Bogosian et al., 1989). They found that norleucine was undesirably incorporated into both native and heterologous proteins being expressed in recombinant strains of E. coli. The level of norleucine incorporation into these proteins ranged from 5% to 15% of the normal methionine content. In this case the norleucine was not being supplied exogenously, but was being naturally synthesized in the E. coli cells. They showed that, in E. coli, the enzymes of the leucine biosynthetic pathway also biosynthesized norleucine, and that the norleucine so formed could be incorporated into protein in place of methionine.
In an effort to produce heterologous proteins with a reduced norleucine content, Bogosian et al. went on to show that the incorporation of norleucine into protein could be reduced by adding additional methionine to the culture medium. They also showed that norleucine biosynthesis could be reduced by supplying exogenous leucine to the culture medium (thereby repressing the induction of leucine biosynthetic enzymes). It was also shown that inactivating one or more of the genes of the leu operon, which encodes the leucine biosynthetic enzymes, prevented the biosynthesis of norleucine (however, a bacterial strain unable to make its own leucine requires the addition of leucine to the culture medium).
Bogosian et al. also demonstrated that the initial substrate for norleucine biosynthesis was 2-ketobutryate, an intermediate in the biosynthesis of isoleucine. Thus, another approach employed by these workers to prevent the biosynthesis of norleucine was to inactivate the ilvA gene. The ilvA gene encodes threonine deaminase, the enzyme that initiates isoleucine biosynthesis by converting threonine to 2-ketobutyrate. However, the ilvA mutant was also incapable of making its own isoleucine. Consequently, this approach necessitated the addition of isoleucine to the culture medium. Thus, while a variety of approaches were devised by these workers to reduce the incorporation of norleucine into protein, they all required the addition of other amino acids (namely, methionine, leucine, or isoleucine) to the culture medium.
Other workers have made similar observations with other heterologous proteins expressed in recombinant E. coli strains. Norleucine was found to be incorporated into human interleukin-2 (Tsai et al. 1988, and Lu et al., 1988), recombinant human insulin-like growth factor I (Forsberg et al., 1990), human macrophage colony stimulating factor (Randhawa, 1994), human leptin (Liu et al., 1997), and human brain-derived neurotrophic factor (Sunasara et al., 1999). With these proteins, norleucine incorporation ranged from 5% to 20% of the normal methionine content.
Since norleucine is not a standard protein amino acid, it is desirable to minimize its incorporation into proteins in order to produce products that are as “natural” as possible (i.e. contain only the amino acids encoded by the DNA sequence). Previously devised methods for reducing the incorporation of norleucine into protein (Tsai et al. 1988, Bogosian et al., 1989, and Randhawa, 1994) were based on the prior art describing the biosynthesis of norleucine and the incorporation of norleucine into protein. That is, the prior art indicated that the biosynthesis of norleucine could be reduced by supplementation of the culture medium with leucine, thereby repressing the enzymes of leucine (and norleucine) biosynthesis. The art also indicated that inactivating the ilvA gene and/or one or more of the genes of the leu operon (namely leuA, leuB, leuC, and leuD) would reduce the biosynthesis of norleucine. Finally, the art indicated that supplementation of the culture medium with methionine would reduce the incorporation of norleucine into protein.
Thus, there are at least two approaches for preventing or reducing the incorporation of norleucine into heterologous proteins described in the existing art discussed above. (1) Inactivation of one or more of the genes encoding the biosynthetic enzymes necessary to produce norleucine. In E. coli, these genes include ilvA, leuA, leuB, leuC, and leuD. (2) Interference with the incorporation of norleucine into protein by supplementing the bacterial growth medium with methionine (or ALIMET® feed supplement, available from Novus International, Inc, St. Louis, Mo., which E. coli can convert into methionine). That is, to competitively block norleucine incorporation into protein using this method, additional methionine accumulates inside the bacteria and competes with the available norleucine for attachment to the methionine tRNA, thereby reducing norleucine incorporation into protein.
Inactivation of one or more of the genes leuA, leuB, leuC, or leuD as a means of reducing norleucine incorporation into protein was also described by Fenton et al., in U.S. Pat. No. 5,599,690. Supplementation of the culture medium with methionine as a means of reducing norleucine incorporation into protein was also described by Fenton et al. in the '690 patent, and by Brunner et al., in U.S. Pat. No. 5,698,418. Brunner et al., in the '418 patent, also provide a description of a means for reducing norleucine incorporation into protein by supplementing the growth medium with other amino acids, specifically, leucine or cysteine. All of these approaches have the disadvantage of requiring the supplementation of the culture medium with one or more amino acids.
Another approach for preventing norleucine incorporation (also described by Brunner. et al. in the '418 patent) is to mutate the protein-encoding gene at the codons originally encoding methionine so that they encode other amino acids. This approach has the disadvantage of altering the primary (and perhaps secondary and tertiary) structure of the protein, which may result in significant and undesirable changes in the biological properties, activity, and usefulness of the protein.
As discussed above, all approaches described, in the existing art, as being effective for reducing the incorporation of norleucine into protein, require either the supplementation of the culture medium with one or more amino acids or the mutation of the gene encoding the protein's amino acid sequence to eliminate methionine codons. It is desirable in the biotechnology industry to be able to cultivate recombinant organisms in a simple chemically defined minimal medium, without the need to add any expensive supplements, such as amino acids while simultaneously reducing the incorporation of norleucine into proteins. Furthermore, it is also desirable to do so without altering the protein's primary amino acid sequence.
Prior to the discovery of the invention disclosed in the instant application, there was no method known in the art that was able to achieve the objective of reducing the incorporation of norleucine into protein without requiring the supplementation of the culture medium with one or more amino acids and/or eliminating the methionine codons from the gene encoding the protein (thereby changing the protein's amino acid sequence).
Norvaline, another non-standard amino acid, is biosynthesized by the same pathway responsible for the synthesis of norleucine (see, Kisumi, et al. (1976) and Bogosian et al. (1989)).
Researchers have shown that, like norleucine, norvaline is sometimes inappropriately incorporated into heterologous proteins. For example, Chiu (1988) and Apostol et al. (1997) reported that norvaline can be incorporated into heterologous proteins, expressed in Escherichia coli, at positions normally occupied by leucine. Similarly Chiu (1988) and Kwong et al. (1998) reported that norvaline can be incorporated in heterologous proteins at positions normally occupied by methionine.
Additionally, other reports indicated that the non-standard amino acids beta-methylnorleucine (Muramatsu et al. (2002)) and homoisoleucine (Sunasara et al. (1999)) are sometimes inappropriately inserted into heterologous proteins, in the place of isoleucine.
Thus, there exists a need for methods of preventing or substantially reducing the incorporation of norleucine, norvaline, beta-methylnorleucine, homoisoleucine, and/or other non-standard amino acids into heterologous proteins. Such a method preferably would not require the use of expensive growth media or amino acid supplements. Neither should the method require alteration of the protein's amino acid sequence; instead the method should result in the incorporation of the proper amino acid into the protein.